RNA interference (RNAi) is a powerful new tool for sequence specific knockdown of targeted mRNAs. The active "trigger" in RNAi is a short double stranded molecule of 21 to 23 nucleotides in length termed small interfering RNAs (siRNAs). SiRNAs can be expressed from promoters in cells either as separate sense and antisense or as short hairpin RNAs (shRNAs). In our hands RNAi is the most powerful inhibitory mechanism for HIV we have utilized, resulting in greater than 4 logs of inhibition of HIV-1 encoded p24 antigen in cell culture. Despite its potency, RNAi can be circumvented by mutations in HIV-1 that disrupt base pairing interactions of the siRNA antisense strand with the target. Since the best anti-HIV-1 therapies incorporate combinatorial drugs maximizing potency and minimizing resistance. In this proposal we will test the hypothesis that combinations of si/shRNAs targeting HIV-1 and the cellular co-receptor CCR5 can provide sustained inhibition of HIV-1 replication in cultured and primary cells. Precursor transcripts encoding several different siRNAs or shRNAs against multiple HIV-1 targets and the cellular CCR5 will be transcribed by Pol III and Pol II promoters. These precursor transcripts will be processed into siRNAs where they function in RNAi. The constructs will be delivered to hematopoietic cells via lentiviral vector transduction. We have already established robust expression of single si/shRNAs in primatry hematopoietic cells, resulting in potent inhibition of HIV-1 replication. It is anticipated that the combinatorial transcripts will provide sustained protection from HIV-1 infection in a genet therapy setting. The proposed research will test several different approaches for expression of combinations of si/shRNAs. The combinatorial constructs will be tested in the context of hematopoiesis both in vitro and in the SCID-hu mouse model. Potent constructs developed here will also be provided to other projects in this program for testing both in T-cells and in the primate stem cell transplant models. The specific aims of this project are: 1) Construction and testing of a combinatorial siRNA genes- A combination of small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) targeting multiple sites in HIV-1 and the CCR5 co-receptor will be co-expressed from a single transcript;2) Testing of Pol Ill versus Pol II promoter systems for expressing combinatorial siRNA constructs- We will compare expression and anti-HIV efficacies of the multi-targeting precursor si and shRNAs using the U6 Pol III promoter, the U1 Pol II promoter and a novel HIV Tat inducible promoter system; 3) SCID-hu mouse studies. Combinatorial constructs from Specific Aim 1 will be transduced into CD34+ hematopoietic precursor cells for anti-viral evaluation in a stem cell setting. Cells will be cultured under conditions that allow differentiation ex vivo into monocytes and macrophages. The capabilities of cells expressing the combinatorial si/shRNAs to form erythroid and myeloid colonies ex vivo will be compared with mock transduced and vector backbone transduced cells. CD34+ cells transduced with combinatorial constructs will also be infused into thy/liv SCID-hu mice. Development of T-lymphocytes will be monitored and compared with vector -transduced cells. Intra-thymic HIV-1 challenges will be carried out to determine the anit-HIV efficacies of combinatorial constructs in an in vivo setting. The long term goal of this research is to provide potent intracellular immunity against HIV-1 in a T-cell and hematopoietic stem cell setting.